CN101117357A - Method for preparing water-soluble beta-1,3/1,6-dextran - Google Patents
Method for preparing water-soluble beta-1,3/1,6-dextran Download PDFInfo
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Abstract
The invention discloses a method for preparing water-soluble beta-1, 3/1, 6-qlucan. The method has the steps as follow: 1) oxidization process: suspending water-insoluble beta-1, 3/1, 6-qlucan in oxidant solution at 2 to 8 DEG C, laying aside for oxidizing reaction for 16 to 24 hours for centrifugation and collection; 2) washing: stirring and washing the deposition treated from step 1) by ethyl alcohol and acetone respectively for 30mins to 1 hour for then centrifugation and collection; 3) ultrasonic process: heavy-density suspending the deposition treated from step 2) in the DMSO solution of concentration expressed in percentage by volume of 0.05 to 0.2 percent, and ultrasonic processing the heavy-density suspension liquid for 20 mins; and centrifugally collecting the supernatant fluid to achieve water-soluble beta-1, 3/1, 6-qlucan. Being detected, the water-soluble beta-1, 3/1, 6-qlucan prepared by the method has the yield over 95 percent and has little protein content and fat content.
Description
Technical field
The present invention relates to a kind of water-soluble beta-1,3/1, the preparation method of 6-dextran.
Background technology
Yeast is a kind of unicellular fungi, is the human up to now microorganism the most extensive, that economic worth is the highest of using.International FDA approval cereuisiae fermentum is the microorganism of safety, widespread use in the production of drink and food.At present, China's year consumes about 2000 tons of yeast, produces about 3 tons waste yeast mud.Because yeast cells wall is the main by product of beer manufacturing industry, and now its main methods is sold as the roughage cheapness or directly discharging exactly, caused very big resource waste and pollution to environment.
Had a plurality of results of study to show, purified yeast cells wall has tangible immunologic enhancement to animal, promotes the growth effect but also show.Further evidence shows that its dominant mechanism is the β-1,3/1 that wherein contains, 6-dextran and mannosans have corresponding immunity and growth-promoting effect, and existing a large amount of experimental study results show β-1,3/1,6-dextran and mannosans are obvious results animal body function regulators.And along with the forbidding gradually of feeding antibiotic, safety, alternative biology feed additive become the industry main flow just gradually.
β-1,3/1, the 6-dextran is the formal formal name used at school of yeast beta-dextran.Its structure is: glucose molecule is with β-1, and the 3-glycosidic link is connected to form main chain, and with β-1, the 6-glycosidic link is connected to form side chain.Glucose molecule sends branch with β-1,6 connection from main chain.
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
-
|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
International and domestic preparation feed immunomodulator β-1,3/1, the main method of 6-dextran is the chemical extraction method.But the β-1,3/1 that adopts chemical process to extract, the yield of 6-dextran is on the low side (＜17%), β-1,3/1, and 6-dextran product purity not high (＜85%) and cost are higher.In the U.S., feed is with β-1,3/1, and the price of 6-dextran probably is 2000～5000 dollars/kilogram (decide on product purity, some even can reach 10000 dollars/kilogram).The main mode of production of mannosans then mostly is microbial fermentation processes, and time-consuming, effort, unstable result (purity＜75%, yield＜15%) and cost are higher.
The β-1,3/1 that directly extracts in the yeast cells wall, the 6-dextran is a water-insoluble beta-1,3/1, the 6-dextran, its extracting method mainly comprises three class methods such as alkaline extraction single stage method, acid extraction single stage method and the compound extraction method of soda acid.
One step of alkali, extraction method was representative with (2003) methods such as suphantharika, and it is intended to optimize extraction process, improves extraction efficiency, takes all factors into consideration the blending ratio of alkali concn, extraction time, extraction temperature and alkaline solution and substrate yeast cells wall.This method adopts progressively determines parameter method, considers each influence factor respectively.Alkali lye), extraction time 1h, temperature be that 90 ℃, alkali concn are 4% (NaOH) drawing each parameter is respectively: blending ratio is that (yeast cells wall:, the yield that obtains product was 19～22% in 1: 5.This method technology is simple, extract yield is higher, protein content is also lower in the product, but β in the extract-1,3/1, the content of 6-dextran is also lower.
One step of the acid method scope of extraction method is wider, mainly is that the kind and the concentration of the acid adopted all alters a great deal.But it is also lower that the main drawback of acid extraction extracts the content of beta-glucan in the product, and reason is more for the beta-glucan of sour institute hydrolysis (degraded) in leaching process.Do not adopt acid extraction method generally speaking.
The method that mainly adopts of various countries mainly concentrated on foundation, improvement and the parameter optimization of soda acid complex method in the last few years.Beta-glucan yield, purity etc. change with technical process a great difference.More representational mainly is the result of Stefan etc. (2003) research, and its technology gentleness, pH do not have strict restriction, and the structure that is intended to keep as far as possible beta-glucan in the cell walls improves extract yield simultaneously.Its extract can get after testing, and the content of beta-glucan is 85%, and seminose is 6%, and protein content is 5%, contains the lipid of trace, and this technology yield can reach 26% of cell walls dry weight.Because its result under pilot plant conditions shows that glucan content also can further improve, mannosans and protein content further descend.
The preparation of water miscible beta-glucan has part results of laboratory report.The insoluble extract that Hunter (2002) obtains alkali, sour compound extraction adopts ultrasonic wave and spray drying technology can prepare the particle that the diameter homogeneous is 1～2 μ M, can be water-soluble.This method can make purer and biological activity better water solubility beta-glucan, but yield lower (total sugar content 85%), removals such as chitin, albumen, fat and nucleic acid not enough (chitin 4.5%, all the other all be between 1～5%)
Summary of the invention
The purpose of this invention is to provide a kind of water-soluble beta-1,3/1, the preparation method of 6-dextran.
A kind of water-soluble beta-1,3/1 provided by the present invention, the preparation method of 6-dextran comprises the steps:
1) oxygenant degradation treatment: with water-insoluble beta-1,3/1, the 6-dextran is suspended in the oxidizing agent solution, at 2～8 ℃, leaves standstill degraded 16～24h, centrifugal collecting precipitation; Described oxidizing agent solution is that to contain concentration expressed in percentage by volume be that 5～15% NaClO, mass percentage concentration are that 0.05～0.2mol/L NaOH and concentration expressed in percentage by volume are the solution of 0.05～0.2% DMSO;
2) washing: the precipitation usefulness ethanol and the acetone of gained in the step 1) are distinguished resuspended agitator treating 30 minutes～1 hour, centrifugal collecting precipitation;
3) supersound process: with step 2) to be resuspended in concentration expressed in percentage by volume be in 0.05～0.2% the DMSO solution, resuspended liquid to be carried out supersound process 20～30 minutes for gained precipitation; Centrifugal collection supernatant liquor obtains water-soluble beta-1,3/1, the 6-dextran solution.
In the described method, in the described step 1), described water-insoluble beta-1,3/1, the concentration that the 6-dextran is suspended in the described oxidizing agent solution is 30～50g/L, is preferably 30g/L; Described oxidizing agent solution is preferably that to contain concentration expressed in percentage by volume be that 10% NaClO, mass percentage concentration 0.1mol/L NaOH and concentration expressed in percentage by volume are the solution of 0.1% DMSO.
In the described method, in the described step 1), the temperature of described oxidizing reaction is 4 ℃, and the time is 24 hours.
In the described method, described step 2) in, the consumption of described ethanol or acetone is a 4-5ml/g precipitation dry weight; Described ethanol or washing with acetone time are preferably 1 hour respectively.
In the described method, in the described step 3), the concentration expressed in percentage by volume of described DMSO solution is preferably 0.1%; The ultrasonic frequency of described supersound process is 20KHz, and ultrasonic power is 50～150W, and be 0.2～0.8s pitch time; Ultrasonic power is preferably 100W, is preferably 0.5s pitch time.
Comprise also in the described method that with described water-soluble beta-1,3/1 the 6-dextran solution concentrates, lyophilize or evaporated under reduced pressure obtain water-soluble beta-1,3/1,6-dextran powder.
In the described method, described water-insoluble beta-1,3/1, the preparation method of 6-dextran comprises the steps:
1. enzymolysis broken wall treatment: add N,O-Diacetylmuramidase in the suspension with yeast cells wall, under 35～40 ℃, enzymolysis, centrifugal collecting precipitation;
2. alkaline purification: the product behind the enzymolysis that 1. step is obtained, in the NaOH of 0.5～1.5mol/L solution, under 75～90 ℃, carried out stir process 2～3 hours, then centrifugal collecting precipitation;
3. acid treatment: the concentration expressed in percentage by volume that is deposited in that 2. step is obtained is in 2～8% the acetic acid solution, under the room temperature condition, and stir process 2～3 hours, centrifugal collecting precipitation;
4. degreasing, remove albumen: with step 3. after the acid treatment the centrifugal precipitation that obtains use the resuspended successively agitator treating of dehydrated alcohol, acetone and ether 30～60 minutes successively, ether left standstill 12～24 hours after handling again, the centrifugal collecting precipitation drying obtains water-insoluble beta-1,3/1, the 6-dextran.
Described step 1. in, described hydrolysis temperature is 37 ℃; Described hydrolysis temperature is preferably 37 ℃.The addition of described N,O-Diacetylmuramidase is that (10000～60000U/g) yeast cells wall dry weights are preferably 1mg/g (20000U/g) yeast cells wall dry weight to 0.5～3mg/g; The concentration of described N,O-Diacetylmuramidase in the suspension of described yeast cells wall is 2000～12000U/ml, is preferably 4000U/ml.Described enzymolysis time is 30～60 minutes, is preferably 30 minutes.
Described step 2. in, the concentration of described NaOH solution is 1mol/L; The temperature of described alkaline purification is 85 ℃, and the alkaline purification time is 3 hours; Product dry weight behind the described enzymolysis is 1g: 5～12ml with the mass/volume of described Na0H solution ratio, is preferably 1g: 5-6ml.
Described step 3. in, the concentration expressed in percentage by volume of described acetic acid solution is 4%; Described acid treatment temperature is a room temperature, and the acid treatment time is 2 hours; The precipitation dry weight that 2. described step obtains is 1g: 5-12ml with the mass/volume of described acetic acid solution ratio, is preferably 1g: 10-11ml; In the described method, also comprise 2., the 2. and 3. middle precipitation of collecting of described step is washed with water.
The present invention combines chemical treatment and ultrasonic disruption, at first adopt the method for chemical oxidation tentatively the macromolecule dextran to be broken into less molecular weight fragment, and then become the dextran of suitable molecular weight size on this basis with ultrasonic disruption, and with the most of non-dextran composition in the organic solvent removal product.Its advantage is to reduce the environmental pollution of simple chemically treated byproduct and the product unhomogeneity of simple ultrasonication, handle the purity and the yield that can improve water-soluble glucan in the product with organic solvent, and reduced the cost that extracts to a certain extent, sum up complete extraction, the purifying process of a cover.
The water-soluble beta-1,3/1 of method of the present invention preparation, 6-dextran after testing, yield can reach more than 95%, protein content and lipid content are very little.The water-soluble beta-1,3/1 of method preparation of the present invention, 6-dextran molecular-weight average is 190KD, is water-soluble saccharan, is the immunomodulator of using in the suitable animal production practice through the animal experiment checking.
Description of drawings
Fig. 1 is a water-insoluble beta-1,3/1, preparation method's schema of 6-dextran.
Fig. 2 is a water-soluble beta-1,3/1, preparation method's schema of 6-dextran.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition described in the following embodiment if no special instructions, is the quality percentage composition.
Embodiment 1, water-soluble beta-1,3/1, the preparation of 6-dextran
One, water-insoluble beta-1,3/1, the preparation of 6-dextran
Water-insoluble beta-1,3/1, the preparation manipulation flow process of 6-dextran as shown in Figure 1, concrete steps are as described below:
1, the enzymolysis of yeast cells wall: (product is available from Yichang Angel Yeast stock company with the 100g yeast cells wall, " Fu Bang " board), 100mg (20000U/mg) N,O-Diacetylmuramidase (is derived from ovalbumin, Cayman) and 500ml water mix (mass ratio is 1000: 1: 5000), use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under 37 ℃ of conditions, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 30 minutes, afterwards with 3000 rev/mins of (Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge, down with) went supernatant to obtain the 95.3g precipitation in centrifugal 20 minutes, be the product behind the yeast cells wall enzymolysis.
2, alkaline purification: with in the adding of the product 95.3g behind the gained parent cell wall enzymolysis in the step 1 500ml 1M NaOH solution (promptly being about the ratio interpolation of 1g: 5ml) according to the mass/volume ratio of product dry weight behind the broken wall and 1M NaOH solution, under 85 ℃ of conditions, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 3 hours, centrifugal 20 minutes with 3600 rev/mins then, obtain supernatant liquor (can continue on for extracting mannosans) and 29.5g precipitation.Precipitation is washed with water three times, obtain the 28.7g precipitation.
3, acid treatment: it is (promptly to be about the ratio interpolation of 1g: 10ml according to the product dry weight after the alkaline purification and the mass/volume ratio of 4% acetic acid solution) in 4% acetic acid solution that the product 28.7g after the gained alkaline purification in the step 2 is added the 300ml concentration expressed in percentage by volume, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory at ambient temperature, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 2 hours, removes supernatant in centrifugal 20 minutes with 3000 rev/mins afterwards.Obtain the 22.2g precipitation, precipitation is washed with water three times.Precipitation is washed with water three times, obtain the 21.5g precipitation.
4, washing with alcohol: the amount of the product after the gained acid treatment in the step 3 (21.5g) according to 0.2-0.25g product (dry weight)/ml joined in the dehydrated alcohol, (18～25 ℃ of room temperatures, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under the condition down together), galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 1 hour, removes supernatant, collecting precipitation in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.
5, washing with acetone: the amount of the precipitated product after the gained washing with alcohol in the step 4 according to 0.2-0.25g product (dry weight)/ml joined in the anhydrous propanone, room temperature condition down stirs (Shanghai Sample Model Factory at a slow speed with agitator at the uniform velocity, galloping horse board adjustable speed agitator, 100～150 rev/mins), churning time is 1 hour, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
6, ether washing: in stink cupboard in join in anhydrous diethyl ether according to the amount of 0.2-0.25g product (dry weight)/ml the precipitated product after the gained acetone treatment in the step 5, room temperature condition is down with the (Shanghai Sample Model Factory at a slow speed of agitator at the uniform velocity, galloping horse board adjustable speed agitator, 100～150 rev/mins) stir, churning time is 1 hour, afterwards container cover lid back room temperature is left standstill 12～24 hours.Then, 2400 rev/mins were removed supernatant in centrifugal 20 minutes, and dry (air-flow dries up or evaporated under reduced pressure or lyophilize) obtains 20.1g water-insoluble beta-1,3/1, the 6-dextran.
7, products therefrom purity adopts total reducing sugar assay method and thin-layer chromatography assay method to determine that the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, and the purity of 6-dextran is 91.5%; Yield adopts product amount/used cell walls amount to compare, and the result is 20.1%; Molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluordetection in cereal extracts.Cereal Chemistry, 80 (4): 485-490) measure, be about 800～850KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
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|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
Protein in the product, fat and coarse ash Determination on content adopt national standard method, are respectively: protein content is 1.15%, lipid content is 0.43%.
Two, water-soluble beta-1,3/1, the preparation of 6-dextran (operating process is as shown in Figure 2)
1, oxide treatment: with the water-insoluble beta-1 of step 1 preparation, 3/1,6-dextran dry powder 20.00g, according to water-insoluble beta-1,3/1,6-dextran: NaClO (concentration expressed in percentage by volume is 10%): NaOH (0.1mol/L): DMSO (concentration expressed in percentage by volume is 0.1%)=1: 10: 10: 10 (g: mL: mL: mL) be made into suspension liquid, at 4 ℃, leave standstill oxidizing reaction 24h in the dark place, after reacting completely, with reaction mixture centrifugal (3000 rev/mins of centrifugal speeds, Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge, down with) collect and obtain the 19.87g precipitation.
2, washing: add in dehydrated alcohol resuspended according to 0.20-0.25g/ml (dry matter basis) centrifugation after the gained oxide treatment in the step 1, room temperature condition is down with average rate agitator middling speed (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins) mix, mixing time is 1 hour, removes supernatant in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.The precipitation that obtains is added in the anhydrous propanone according to 0.20-0.25g/ml (dry matter basis), room temperature condition is used average rate agitator (Shanghai Sample Model Factory at a slow speed down, galloping horse board adjustable speed agitator, 100～150 rev/mins) mix, mix 1 hour time, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
3, supersound process: it is in 0.1% the DMSO solution that step 2 gained precipitation is resuspended in concentration expressed in percentage by volume, with resuspended liquid carry out ultrasonication (ultrasonic time 20min, ultrasonic frequency 20KHz, ultrasonic power 100W, pitch time 0.5s).
4, behind ultrasonic the finishing with mixture centrifugal precipitation of removing under 3000rpm, concentrated supernatant, lyophilize or evaporated under reduced pressure obtain 19.19g water-soluble beta-1,3/1, the 6-dextran.
5, the products therefrom molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluordetection in cereal extracts.Cereal Chemistry, 80 (4): 485-490) measure, molecular-weight average is 190KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-G-(1，3)-β-D-G-(1，3)-β-D-G-(1，3)-β-D-G-
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|1，6
β-D-G-(1，6)-β-D-Quip-(1，6)
Yield can reach 95.95%.The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.15%, lipid content is 0.43%.
Embodiment 2, water-soluble beta-1,3/1, the preparation of 6-dextran
One, water-insoluble beta-1,3/1, the preparation of 6-dextran
Water-insoluble beta-1,3/1, the preparation manipulation flow process of 6-dextran as shown in Figure 1, concrete steps are as described below:
1, the enzymolysis of yeast cells wall: 200g yeast cells wall, 400mg (20000U/mg) N,O-Diacetylmuramidase (are derived from ovalbumin, Cayman) and 500ml water mix, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under 35 ℃ of conditions, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 60 minutes, afterwards with 3000 rev/mins of (Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge) goes supernatant to obtain 191.20g precipitation in centrifugal 25 minutes, be the product behind the yeast cells wall enzymolysis.
2, alkaline purification: with in the adding of the product 191.20g behind the gained yeast cells wall enzymolysis in the step 1 2000ml 0.5M NaOH solution (promptly being about the ratio interpolation of 1g: 10ml) according to the mass/volume ratio of product dry weight behind the broken wall and NaOH solution, using at the uniform velocity under 75 ℃ of conditions, the agitator middling speed stirs (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins), churning time is 3 hours, centrifugal 20 minutes with 3600 rev/mins then, obtain supernatant liquor 2040ml and continue on for extracting mannosans, (be used to extract water-insoluble beta-1 with 50.4 g precipitation, 3/1, the 6-dextran).
3, acid treatment: it is (promptly to be about the ratio interpolation of 1g: 5ml according to the product dry weight after the alkaline purification and the mass/volume ratio of acetic acid solution) in 2% acetic acid solution that the product 50.4g after the gained alkaline purification in the step 2 is added the 260ml volumn concentration, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory at ambient temperature, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 2 hours, removes supernatant in centrifugal 20 minutes with 3000 rev/mins afterwards.Obtain the 36.12g precipitation, precipitation is washed with water three times.Precipitation is washed with water three times, obtain the 35.80g precipitation.
4, washing with alcohol: the amount of the product after the gained acid treatment in the step 3 (35.80g) according to 0.2-0.25g product (dry weight)/ml joined in the dehydrated alcohol, using at the uniform velocity under room temperature (18～25 ℃) condition, the agitator middling speed stirs, churning time is 30 minutes, removed supernatant, collecting precipitation in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.
5, washing with acetone: the amount of the precipitated product after the gained washing with alcohol in the step 4 according to 0.2-0.25g product (dry weight)/ml joined in the anhydrous propanone, stir (Shanghai Sample Model Factory at a slow speed with agitator at the uniform velocity under room temperature (18～25 ℃) condition, galloping horse board adjustable speed agitator, 100～150 rev/mins), churning time is 30 minutes, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
6, ether washing: in stink cupboard in join in anhydrous diethyl ether according to the amount of 0.2-0.25g product (dry weight)/ml the precipitated product after the gained acetone treatment in the step 5, under room temperature (18～25 ℃) condition with the (Shanghai Sample Model Factory at a slow speed of agitator at the uniform velocity, galloping horse board adjustable speed agitator, 100～150 rev/mins) stir, churning time is 30 minutes, afterwards container cover lid back room temperature is left standstill 12～24 hours.Then, 2400 rev/mins were removed supernatant in centrifugal 20 minutes, and dry (air-flow dries up or evaporated under reduced pressure or lyophilize) obtains 34.63g water-insoluble beta-1,3/1, the 6-dextran.
7, products therefrom purity adopts total reducing sugar assay method and thin-layer chromatography assay method to determine, is 89.51%; Yield adopts product amount/used cell walls amount to compare, and the result is 17.31%; Molecular weight is according to improving fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecularweight using SEC with calcofluor detection in cereal extracts.CerealChemistry, 80 (4): 485-490) measure, the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, the molecular weight of 6-dextran is 800～850KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
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|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.14%, lipid content is 0.52%.
Two, water-soluble beta-1,3/1, the preparation of 6-dextran (operating process is as shown in Figure 2)
1, oxide treatment: with the water-insoluble beta-1 of step 1 preparation, 3/1,6-dextran dry powder 20.00g, according to water-insoluble beta-1,3/1,6-dextran: NaClO (concentration expressed in percentage by volume is 5%): NaOH (0.05mol/L): DMSO (concentration expressed in percentage by volume is 0.05%)=1: 10: 10: 10 (g: mL: mL: mL) be made into suspension liquid, at 4 ℃, leave standstill oxidizing reaction 24h in the dark place, after reacting completely, with reaction mixture centrifugal (3000 rev/mins of centrifugal speeds, Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge, down with) collect and obtain the 19.88g precipitation.
2, washing: add in dehydrated alcohol resuspended according to 0.20-0.25g/ml (dry matter basis) centrifugation after the gained oxide treatment in the step 1, room temperature condition is down with average rate agitator middling speed (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins) mix, mixing time is 30 hours, removes supernatant in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.The precipitation that obtains is added in the anhydrous propanone according to 0.20-0.25g/ml (dry matter basis), room temperature condition is used average rate agitator (Shanghai Sample Model Factory at a slow speed down, galloping horse board adjustable speed agitator, 100～150 rev/mins) mix, mix 1 hour time, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
3, supersound process: it is in 0.05% the DMSO solution that step 2 gained precipitation is resuspended in concentration expressed in percentage by volume, with resuspended liquid carry out ultrasonication (ultrasonic time 20min, ultrasonic frequency 20KHz, ultrasonic power 50W, pitch time 0.2s).
4, behind ultrasonic the finishing with mixture centrifugal precipitation of removing under 3000rpm, concentrated supernatant, lyophilize or evaporated under reduced pressure obtain 18.13g water-soluble beta-1,3/1, the 6-dextran.
5, the products therefrom molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluordetection in cereal extracts.Cereal Chemistry, 80 (4): 485-490) measure, molecular-weight average is 190KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-G-(1，3)-β-D-G-(1，3)-β-D-G-(1，3)-β-D-G-
-
|1，6
β-D-G-(1，6)-β-D-Quip-(1，6)
Yield can reach 90.2%.The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.17%, lipid content is 0.42%.
Embodiment 3, water-soluble beta-1,3/1, the preparation of 6-dextran
One, water-insoluble beta-1,3/1, the preparation of 6-dextran
Water-insoluble beta-1,3/1, the preparation manipulation flow process of 6-dextran as shown in Figure 1, concrete steps are as described below:
1, the enzymolysis of yeast cells wall: 200g yeast cells wall, 600mg (20000U/mg) N,O-Diacetylmuramidase (are derived from ovalbumin, Cayman) and 1000ml water mix, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory under 40 ℃ of conditions, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 30 minutes, afterwards with 3000 rev/mins of (Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge) goes supernatant to obtain 190.8g precipitation in centrifugal 20 minutes, be the product behind the yeast cells wall enzymolysis.
2, alkaline purification: with in the adding of the product 190.8g behind the gained yeast cells wall enzymolysis in the step 1 2000ml 1.0M NaOH solution (promptly being about the ratio interpolation of 1g: 10ml) according to the mass/volume ratio of product dry weight behind the broken wall and NaOH solution, using at the uniform velocity under 90 ℃ of conditions, the agitator middling speed stirs (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins), churning time is 2.5 hours, centrifugal 20 minutes with 3600 rev/mins then, obtain supernatant liquor 2040ml and continue on for extracting mannosans, (be used to extract water-insoluble beta-1 with the 57.4g precipitation, 3/1, the 6-dextran).
3, acid treatment: it is (promptly to be about the ratio interpolation of 1g: 10ml according to the product dry weight after the alkaline purification and the mass/volume ratio of acetic acid solution) in 8% acetic acid solution that the product 57.4g after the gained alkaline purification in the step 2 is added the 600ml volumn concentration, use at the uniform velocity agitator middling speed (Shanghai Sample Model Factory at ambient temperature, galloping horse board adjustable speed agitator, 200～300 rev/mins) stir, churning time is 2.5 hours, removes supernatant in centrifugal 20 minutes with 3000 rev/mins afterwards.Obtain the 40.2g precipitation, precipitation is washed with water three times.Precipitation is washed with water three times, obtain the 38.0g precipitation.
4, washing with alcohol: the amount of the product after the gained acid treatment in the step 3 (38.0g) according to 0.2-0.25g product (dry weight)/ml joined in the dehydrated alcohol, using at the uniform velocity under room temperature (18～25 ℃) condition, the agitator middling speed stirs, churning time is 50 minutes, removed supernatant, collecting precipitation in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.
5, washing with acetone: the amount of the precipitated product after the gained washing with alcohol in the step 4 according to 0.2-0.25g product (dry weight)/ml joined in the anhydrous propanone, stir (Shanghai Sample Model Factory at a slow speed with agitator at the uniform velocity under room temperature (18～25 ℃) condition, galloping horse board adjustable speed agitator, 100～150 rev/mins), churning time is 40 minutes, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
6, ether washing: in stink cupboard in join in anhydrous diethyl ether according to the amount of 0.2-0.25g product (dry weight)/ml the precipitated product after the gained acetone treatment in the step 5, under room temperature (18～25 ℃) condition with the (Shanghai Sample Model Factory at a slow speed of agitator at the uniform velocity, galloping horse board adjustable speed agitator, 100～150 rev/mins) stir, churning time is 30 minutes, afterwards container cover lid back room temperature is left standstill 12～24 hours.Then, 2400 rev/mins were removed supernatant in centrifugal 20 minutes, and dry (air-flow dries up or evaporated under reduced pressure or lyophilize) obtains 37.0g water-insoluble beta-1,3/1, the 6-dextran.
7, products therefrom purity adopts total reducing sugar assay method and thin-layer chromatography assay method to determine, is 90.37%; Yield adopts product amount/used cell walls amount to compare, and the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, and the yield of 6-dextran is 18.54%; Molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluordetection in cereal extracts.Cereal Chemistry, 80 (4): 485-490) measure, the result shows the above-mentioned water-insoluble beta that obtains-1,3/1, the molecular weight of 6-dextran is 800～850KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-Glcp-(1，3)-β-D-GlcpNAc-
-
|1，6
β-D-Glcp-(1，6)-β-D-Quip-(1，6)
The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.04%, lipid content is 0.40%.
Two, water-soluble beta-1,3/1, the preparation of 6-dextran (operating process is as shown in Figure 2)
1, oxide treatment: with the water-insoluble beta-1 of step 1 preparation, 3/1,6-dextran dry powder 20.00g, according to water-insoluble beta-1,3/1,6-dextran: NaClO (concentration expressed in percentage by volume is 15%): NaOH (0.2mol/L): DMSO (concentration expressed in percentage by volume is 0.2%)=1: 10: 10: 10 (g: mL: mL: mL) be made into suspension liquid, at 4 ℃, leave standstill oxidizing reaction 24h in the dark place, after reacting completely, with reaction mixture centrifugal (3000 rev/mins of centrifugal speeds, Anting Scientific Instrument Factory, Shanghai, DL6000B type refrigerated centrifuge, down with) collect and obtain the 19.87g precipitation.
2, washing: add in dehydrated alcohol resuspended according to 0.20-0.25g/ml (dry matter basis) centrifugation after the gained oxide treatment in the step 1, room temperature condition is down with average rate agitator middling speed (Shanghai Sample Model Factory, galloping horse board adjustable speed agitator, 200～300 rev/mins) mix, mixing time is 30 hours, removes supernatant in centrifugal 20 minutes with 3000 rev/mins of speed afterwards.The precipitation that obtains is added in the anhydrous propanone according to 0.20-0.25g/ml (dry matter basis), room temperature condition is used average rate agitator (Shanghai Sample Model Factory at a slow speed down, galloping horse board adjustable speed agitator, 100～150 rev/mins) mix, mix 1 hour time, removed supernatant, collecting precipitation in centrifugal 20 minutes with 2400 rev/mins of speed afterwards.
3, supersound process: it is in 0.2% the DMSO solution that step 2 gained precipitation is resuspended in concentration expressed in percentage by volume, with resuspended liquid carry out ultrasonication (ultrasonic time 20min, ultrasonic frequency 20KHz, ultrasonic power 150W, pitch time 0.8s).
4, behind ultrasonic the finishing with mixture centrifugal precipitation of removing under 3000rpm, concentrated supernatant, lyophilize or evaporated under reduced pressure obtain 18.45g water-soluble beta-1,3/1, the 6-dextran.
5, the products therefrom molecular weight is according to fluorescent method (Rinsten, L., T.Stenberg and R.Andersson.2003.Determination of β-glucan molecular weight using SEC with calcofluordetection in cereal extracts.Cereal Chemistry, 80 (4): 485-490) measure, molecular-weight average is 190KD.Structure detection is as follows through infrared spectra and nuclear magnetic resonance spectroscopy:
-「-β-D-G-(1，3)-β-D-G-(1，3)-β-D-G-(1，3)-β-D-G-
-
|1，6
β-D-G-(1，6)-β-D-Quip-(1，6)
Yield can reach 92.25%.The protein of product and lipid content adopt national standard method to carry out, and are respectively: protein content is 1.19%, lipid content is 0.47%.
Claims (10)
1. water-soluble beta-1,3/1, the preparation method of 6-dextran comprises the steps:
1) oxide treatment: with water-insoluble beta-1,3/1, the 6-dextran is suspended in the oxidizing agent solution, at 2～8 ℃, leaves standstill oxidizing reaction 16～24h, centrifugal collecting precipitation; Described oxidizing agent solution is that to contain concentration expressed in percentage by volume be that 5～15% NaClO, mass percentage concentration are that 0.05～0.2mol/L NaOH and concentration expressed in percentage by volume are the solution of 0.05～0.2% DMSO;
2) washing: the precipitation usefulness ethanol and the acetone of gained in the step 1) are distinguished resuspended agitator treating 30 minutes～1 hour, centrifugal collecting precipitation;
3) supersound process: with step 2) to be resuspended in concentration expressed in percentage by volume be in 0.05～0.2% the DMSO solution, resuspended liquid to be carried out supersound process 20～30 minutes for gained precipitation; Centrifugal collection supernatant liquor obtains water-soluble beta-1,3/1, the 6-dextran solution.
2. method according to claim 1 is characterized in that: in the described step 1), and described water-insoluble beta-1,3/1, the concentration that the 6-dextran is suspended in the described oxidizing agent solution is 30～50g/L, is preferably 30g/L; Described oxidizing agent solution is that to contain concentration expressed in percentage by volume be that 10% NaClO, mass percentage concentration 0.1mol/L NaOH and concentration expressed in percentage by volume are the solution of 0.1% DMSO.
3. method according to claim 2 is characterized in that: in the described step 1), the temperature of described oxidizing reaction is 4 ℃, and the time is 24 hours.
4. method according to claim 3 is characterized in that: described step 2), the consumption of described ethanol or acetone is a 4-5ml/g precipitation dry weight; Described ethanol or washing with acetone time were respectively 1 hour.
5. method according to claim 4 is characterized in that: in the described step 3), the concentration expressed in percentage by volume of described DMSO solution is 0.1%; The condition of described supersound process is that ultrasonic frequency is 20KHz, and ultrasonic power is 50～150W, and be 0.2～0.8s pitch time; Described ultrasonic power is preferably 100W, is preferably 0.5s pitch time.
6. method according to claim 5 is characterized in that: comprise also in the described method that with described water-soluble beta-1,3/1 the 6-dextran solution concentrates, lyophilize or evaporated under reduced pressure obtain water-soluble beta-1,3/1,6-dextran powder.
7. according to any among the claim 1-6-described method, it is characterized in that: described water-insoluble beta-1,3/1, the preparation method of 6-dextran comprises the steps:
1. enzymolysis broken wall treatment: add N,O-Diacetylmuramidase in the suspension with yeast cells wall, under 35～40 ℃, enzymolysis, centrifugal collecting precipitation;
2. alkaline purification: the product behind the enzymolysis that 1. step is obtained, in the NaOH of 0.5～1.5mol/L solution, under 75～90 ℃, carried out stir process 2～3 hours, then centrifugal collecting precipitation;
3. acid treatment: the concentration expressed in percentage by volume that is deposited in that 2. step is obtained is in 2～8% the acetic acid solution, under the room temperature condition, and stir process 2～3 hours, centrifugal collecting precipitation;
4. degreasing, remove albumen: with step 3. after the acid treatment the centrifugal precipitation that obtains with dehydrated alcohol, acetone and ether resuspended successively agitator treating 30 minutes～1 hour respectively, ether left standstill 12～24 hours after handling again, the centrifugal collecting precipitation drying obtains water-insoluble beta-1,3/1, the 6-dextran.
8. method according to claim 7 is characterized in that: described step 1. in, described hydrolysis temperature is 37 ℃; The addition of described N,O-Diacetylmuramidase is 20000～60000U/g yeast cells wall dry weight, is preferably 20000U/g yeast cells wall dry weight; The concentration of described N,O-Diacetylmuramidase in the suspension of described yeast cells wall is 4000～12000U/ml, is preferably 4000U/ml; Described enzymolysis time is 20～60 minutes, is preferably 30 minutes.
9. method according to claim 8 is characterized in that: described step 2. in, the concentration of described NaOH solution is 1mol/L; The temperature of described alkaline purification is 85 ℃, and the alkaline purification time is 3 hours; Product dry weight behind the described enzymolysis is 1g: 5～12ml with the mass/volume of described NaOH solution ratio, is preferably 1g: 5-6ml.
10. method according to claim 9 is characterized in that: described step 3. in, the concentration expressed in percentage by volume of described acetic acid solution is 4%; Described acid treatment temperature is 37 ℃, and the acid treatment time is 2 hours; The precipitation dry weight that 2. described step obtains is 1g: 5～12ml with the mass/volume of described acetic acid solution ratio, is preferably 1g: 11ml; In the described method, also comprise 2., the 2. and 3. middle precipitation of collecting of described step is washed with water.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101353383B (en) * | 2008-09-17 | 2010-12-08 | 山东京博控股发展有限公司 | Water-soluble yeast beta-dextran and preparation thereof |
| CN105925640A (en) * | 2016-05-16 | 2016-09-07 | 上海艾苛密进出口有限公司 | Preparation method of yeast beta-glucan |
| CN106755196A (en) * | 2016-12-20 | 2017-05-31 | 广东工业大学 | One kind improves the water miscible method of beta glucan |
| CN106905442A (en) * | 2017-03-16 | 2017-06-30 | 山东大学齐鲁医院 | A kind of preparation method for improving the glucans of small molecule β 1,3 of hepatitis immunity |
| CN107148431A (en) * | 2014-10-31 | 2017-09-08 | 温特沙尔控股有限公司 | Method for concentrating beta glucan |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE19911056B9 (en) * | 1999-03-12 | 2005-09-08 | Biotec Asa | Cosmetic preparations and their use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101353383B (en) * | 2008-09-17 | 2010-12-08 | 山东京博控股发展有限公司 | Water-soluble yeast beta-dextran and preparation thereof |
| CN107148431A (en) * | 2014-10-31 | 2017-09-08 | 温特沙尔控股有限公司 | Method for concentrating beta glucan |
| CN105925640A (en) * | 2016-05-16 | 2016-09-07 | 上海艾苛密进出口有限公司 | Preparation method of yeast beta-glucan |
| CN105925640B (en) * | 2016-05-16 | 2019-10-11 | 上海艾苛密进出口有限公司 | The preparation method of yeast beta-dextran |
| CN106755196A (en) * | 2016-12-20 | 2017-05-31 | 广东工业大学 | One kind improves the water miscible method of beta glucan |
| CN106905442A (en) * | 2017-03-16 | 2017-06-30 | 山东大学齐鲁医院 | A kind of preparation method for improving the glucans of small molecule β 1,3 of hepatitis immunity |
| CN109797179A (en) * | 2019-03-20 | 2019-05-24 | 常州市第二人民医院 | A kind of environment-friendly preparation method thereof of yeast dextran |
| CN113230389A (en) * | 2021-04-29 | 2021-08-10 | 意润健康产业（广州）有限公司 | Application of composition comprising enzymolysis ovalbumin micromolecule active short peptide in preparation of medicines and foods for improving hypoalbuminemia |
| CN113307894A (en) * | 2021-07-01 | 2021-08-27 | 上海应用技术大学 | Method for solubilizing yeast beta-glucan by ultrasonic-assisted hydrogen peroxide method |
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